David I. Hughes

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid‐lumbar spinal cord of the rat. The great majority (94–100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2‐immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin‐ and enkephalin‐containing boutons (including somatostatin‐immunoreactive boutons that lacked calcitonin gene‐related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present.

Physiological and morphological diversity of immunocytochemically defined parvalbumin- and cholecystokinin-positive interneurones in CA1 of the adult rat hippocampus

To investigate the electrophysiological properties, synaptic connections, and anatomy of individual parvalbumin‐immunoreactive (PV‐IR) and cholecystokinin‐immunoreactive (CCK‐IR) interneurones in CA1, dual intracellular recordings using biocytin‐filled microelectrodes in slices of adult rat hippocampus were combined with fluorescence labelling of PV‐ and CCK‐containing cells. Of 36 PV‐IR cells, 29 were basket cells, with most of their axonal arbours in the stratum pyramidale (SP). Six were bistratified cells with axons ramifying throughout stratum oriens (SO) and stratum radiatum (SR). One was a putative axo‐axonic cell with an axonal arbour confined to half of the SP and a narrow adjacent region of the SO. Of 27 CCK‐IR neurones, 13 were basket cells, with most of their axonal arbours in the SP, and included basket cells with somata in the SP (6), SO (3), and SR (2) and at the border between the stratum lacunosum‐moleculare (SLM) and the SR (2). In addition, several dendrite‐targeting cell classes expressed CCK‐IR: 4 of 9 bistratified cells with axons ramifying in the SO and SR; all five Schaffer‐associated cells whose axons ramified extensively in the SR; both cells classified as quadrilaminar because their axons ramified in the SO, SP, SR, and SLM; one SO‐SO cell whose dendritic and axonal arbours were contained within the SO; and one perforant path‐associated cell with axonal and dendritic arbours within the distal SR and SLM. The majority (31 of 36) of PV‐IR neurones recorded were fast‐spiking, and most fast‐spiking cells tested (25 of 29 basket, 1 axo‐axonic, and 5 of 6 bistratified cells) were PV‐IR. However, 1 of 6 regular‐spiking basket, 1 of 4 regular‐spiking bistratified, and 3 of 5 burst‐firing basket cells were also PV‐IR. In contrast, the majority (17 of 27) of the CCK‐IR neurones recorded were regular‐spiking, 3 were burst‐firing, and 7 were fast‐spiking. These data confirm that the majority of PV‐IR and CCK‐IR axon terminals innervate proximal portions of CA1 pyramidal cells. Most PV‐IR cells are fast‐spiking, whereas most CCK‐IR cells are regular‐spiking. In both neurochemical classes basket cells predominate, but both groups included subpopulations of dendrite‐targeting cells. Despite these similarities, the two populations exhibited very different somatic distributions, and each contained cellular morphologies not represented in the other. J. Comp. Neurol. 443:346–367, 2002.

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain.

&NA; GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABAA and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA‐immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA‐ and/or glycine‐immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham‐operated and naïve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I–III of the ipsilateral dorsal horn that showed GABA‐ or glycine‐immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham‐operated or naïve animals. In addition, we did not see any evidence for alterations of GABA‐ or glycine‐immunostaining in the neuropil of laminae I–III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach.

&NA; Lamina II contains a large number of interneurons involved in modulation and transmission of somatosensory (including nociceptive) information. However, its neuronal circuitry is poorly understood due to the difficulty of identifying functional populations of interneurons. This information is important for understanding nociceptive processing and for identifying changes that underlie chronic pain. In this study, we compared morphology, neurotransmitter content, electrophysiological and pharmacological properties for 61 lamina II neurons recorded in slices from adult rat spinal cord. Morphology was related to transmitter content, since islet cells were GABAergic, while radial and most vertical cells were glutamatergic. However, there was considerable diversity among the remaining cells, some of which could not be classified morphologically. Transmitter phenotype was related to firing pattern, since most (18/22) excitatory cells, but few (2/23) inhibitory cells had delayed, gap or reluctant patterns, which are associated with A‐type potassium (IA) currents. Somatostatin was identified in axons of 14/24 excitatory neurons. These had variable morphology, but most of those tested showed delayed‐firing. Excitatory interneurons are therefore likely to contribute to pain states associated with synaptic plasticity involving IA currents. Although noradrenaline and serotonin evoked outward currents in both inhibitory and excitatory cells, somatostatin produced these currents only in inhibitory neurons, suggesting that its pro‐nociceptive effects are mediated by disinhibition. Our results demonstrate that certain distinctive populations of inhibitory and excitatory interneuron can be recognised in lamina II. Combining this approach with identification of other neurochemical markers should allow further clarification of neuronal circuitry in the superficial dorsal horn.

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord.

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

Loss of Neurons from Laminas I-III of the Spinal Dorsal Horn Is Not Required for Development of Tactile Allodynia in the Spared Nerve Injury Model of Neuropathic Pain

It has been proposed that death of inhibitory interneurons in the dorsal horn contributes to the neuropathic pain that follows partial nerve injury. In this study, we have used two approaches to test whether there is neuronal death in the dorsal horn in the spared nerve injury (SNI) model. We performed a stereological analysis of the packing density of neurons in laminas I-III 4 weeks after operation and found no reduction on the ipsilateral side compared with that seen on the contralateral side or in sham-operated or naive rats. In addition, we used two markers of apoptosis, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and immunocytochemical detection of cleaved (activated) caspase-3. Neither of these methods demonstrated apoptotic neurons in the dorsal spinal cord 1 week after operation. Although TUNEL-positive cells were present throughout the gray and white matter at this stage, they were virtually all labeled with antibody against ionized calcium-binding adapter molecule 1, a marker for microglia. All animals that underwent SNI showed clear signs of tactile allodynia affecting the ipsilateral hindpaw. These results suggest that a significant loss of neurons from the dorsal horn is not necessary for the development of tactile allodynia in the SNI model.

Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus.

Hippocampal pyramidal cells express several α‐subunits, which determine the affinity of GABAA (γ‐aminobutyric acid) receptors for benzodiazepine site ligands. This study asked whether inhibitory postsynaptic potentials (IPSPs) elicited by specific interneuronal subclasses were differentially sensitive to the α1‐preferring agonist Zolpidem, i.e. whether different receptors mediate different inhibitory connections. Paired intracellular recordings in which the presynaptic cell was an interneuron and the postsynaptic cell a CA1 pyramid were performed in slices of adult rat hippocampus. Resultant IPSPs were challenged with Zolpidem, cells filled with biocytin and identified morphologically. IPSPs elicited by fast spiking (FS) basket cells (n = 9) were enhanced more than IPSPs elicited by regular spiking (RS) basket cells (n = 10). At FS basket cell synapses the efficacy of Zolpidem was equivalent to that of Diazepam, while RS basket cell IPSPs are enhanced 50% less by Zolpidem than by Diazepam. Thus, while α1 subunits may dominate at synapses supplied by FS basket cells, RS basket cell synapses also involve α2/3 subunits. Two bistratified cell IPSPs tested with Zolpidem did not increase in amplitude, despite powerful enhancements of bistratified cell IPSPs by Diazepam, consistent with previous indications that these synapses utilize α5‐containing receptors. Enhancements of basket cell IPSPs by Zolpidem and Diazepam were bi‐ or triphasic with steep amplitude increases separated by plateaux, occurring 10–15, 25–30 and 45–55 min after adding the drug to the bath. The entire enhancement was, however, blocked by the antagonist Flumazenil (n = 7). Flumazenil, either alone (n = 3), or after Zolpidem, reduced IPSP amplitude to ∼ 90% of control, suggesting that α4‐containing receptors were not involved.

Morphological, neurochemical and electrophysiological features of parvalbumin‐expressing cells: a likely source of axo‐axonic inputs in the mouse spinal dorsal horn

•  Perception of normal bodily sensations relies on the precise regulation of sensory information entering the dorsal horn of the spinal cord. •  Inhibitory, axoaxonic, synapses provide a mechanism for this regulation, but the source of these important inhibitory connections remains to be elucidated. •  This study shows that a subpopulation of spinal interneurons that expresses parvalbumin and have specific morphological, connectivity and functional characteristics are a likely source of the inhibitory inputs that selectivity regulate non‐noxious tactile input in the spinal cord. •  Our findings suggest that a loss of normal function in parvalbumin positive dorsal horn neurons may result in the development of tactile allodynia, where non‐painful stimuli gain the capacity to evoke the sensation of pain.

Peripheral axotomy induces depletion of the vesicular glutamate transporter VGLUT1 in central terminals of myelinated afferent fibres in the rat spinal cord.

Myelinated primary afferent axons use glutamate as their principal neurotransmitter. We have shown previously that central terminals of myelinated tactile and proprioceptive afferents contain the vesicular glutamate transporter VGLUT1. Peripheral nerve injury is known to induce changes in the anatomy, neurochemistry, and physiology of primary afferents. In this study, we have examined the effect of peripheral axotomy on VGLUT1 expression in central terminals of myelinated afferents in laminae III-V and lamina IX of the rat spinal cord. Bilateral injections of cholera toxin B subunit (CTb) were made into the sciatic nerves of rats that had undergone unilateral sciatic nerve transection 1, 2, 4, or 8 weeks previously. Immunofluorescence staining and confocal microscopy were used to compare levels of VGLUT1 in CTb-labelled boutons on the intact and sectioned sides at each postoperative survival time. VGLUT1 was depleted from central terminals of transected myelinated afferents in rats injected with CTb 1 week after nerve section, and this depletion became more severe in animals with longer postaxotomy survival times. By 4 weeks, the level of VGLUT1 in CTb-labelled boutons in lamina IX was reduced by over 80% compared to that seen in intact (contralateral) afferents, while for boutons in laminae III-V, VGLUT1 levels were reduced by 50-70%. This suggests that loss of VGLUT1 is more severe in proprioceptive than cutaneous afferents. Depletion of VGLUT1 may lead to a decrease in levels of transmitter glutamate in these afferents and thus to a reduction in synaptic efficacy.

A Quantitative Study of Inhibitory Interneurons in Laminae I-III of the Mouse Spinal Dorsal Horn

Laminae I-III of the spinal dorsal horn contain many inhibitory interneurons that use GABA and/or glycine as a neurotransmitter. Distinct neurochemical populations can be recognised among these cells, and these populations are likely to have differing roles in inhibiting pain or itch. Quantitative studies in rat have shown that inhibitory interneurons account for 25-40% of all neurons in this region. The sst2A receptor is expressed by around half the inhibitory interneurons in laminae I-II, and is associated with particular neurochemically-defined populations. Although much of the work on spinal pain mechanisms has been performed on rat, the mouse is now increasingly used as a model, due to the availability of genetically altered lines. However, quantitative information on the arrangement of interneurons is lacking in the mouse, and it is possible that there are significant species differences in neuronal organisation. In this study, we show that as in the rat, nearly all neurons in laminae I-III that are enriched with glycine also contain GABA, which suggests that GABA-immunoreactivity can be used to identify inhibitory interneurons in this region. These cells account for 26% of the neurons in laminae I-II and 38% of those in lamina III. As in the rat, the sst2A receptor is only expressed by inhibitory interneurons in laminae I-II, and is present on just over half (54%) of these cells. Antibody against the neurokinin 1 receptor was used to define lamina I, and we found that although the receptor was concentrated in this lamina, it was expressed by many fewer cells than in the rat. By estimating the total numbers of neurons in each of these laminae in the L4 segment of the mouse, we show that there are around half as many neurons in each lamina as are present in the corresponding segment of the rat.